1. Field
The invention is in the field of capillary electrophoresis, detection methods for capillary electrophoresis and apparatus based on such methods, and on Schlieren optics.
2. State of the Art
It has been known for some time that a refractive index gradient such as produced by a concentration gradient in a fluid such as a gas, liquid, or supercritical fluid, will cause deflection of light passed through the gradient. The optical method of observing and measuring the deflection of light caused by refractive index gradient fields is generally referred to as Schlieren optics. In the past, Schlieren images resulting from light deflections have been recorded on photographic plates and the plates then analyzed for light intensity distribution using densitometers. Recently, evaluation of the photographic images has been done by computer. These methods are useful in studying plasmas where very complicated toroidal and parabolic shapes are generated.
U.S. Pat. No. 4,547,071 discloses a sensor for measuring density gradients in a nonhomogeneous fluid sample using Schlieren optics. In such sensor, a laser light beam is directed through a sample chamber and is moved along said chamber. A quadrant light position sensor located on the opposite side of the chamber detects the deflection of the laser light beam as it is moved through the sample. The amount of deflection indicates the density gradient at any point in the sample. Rather than moving the laser beam along the sample chamber, the beam can be held constant and the sample chamber with sample therein moved. However, moving a laser and detector together in relation to a sample chamber and keeping the laser beam focused on the sample chamber, even a relatively large chamber, is difficult, as is moving a sample chamber through the laser beam so as to keep the laser beam properly focused. Trying to do either with a small capillary sample chamber is very difficult and impractical.
My U.S. Pat. Nos. 4,784,494, 4,940,333 and 4,993,832 show detectors that can be used to detect concentration and thermal gradients in very small samples. The detectors utilize a light source to generate one or two probe beams of light that pass through the sample having the gradient to be detected and the deflection of the probe beam or beams is measured on a beam position detector. Various light sources may be used to generate the probe beam or beams, such as a laser or light emitting diode (LED). These detectors, however, are designed generally to be used where the gradients to be detected move through the probe beam or beams of light.
Capillary electrophoresis has become an important separation method in bioanalytical chemistry. Separation and detection of very small amounts of biological samples, about pL-nL volumes, can be achieved with capillary electrophoresis. This is generally not possible with more conventional methods of separation, even with high performance liquid chromatography. There are several capillary electrophoresis separation methods in use for different kinds of samples. They include capillary zone electrophoresis, moving boundary capillary electrophoresis, capillary isotachophoresis, and capillary isoelectric focusing. Capillary zone electrophoresis, moving boundary capillary electrophoresis, and isotachophoresis all have the advantage that the sample moves through a capillary sample separation chamber during the separation so can be used with the detectors of my cited prior patents. Capillary zone electrophoresis and moving boundary capillary electrophoresis are dynamic processes where separation occurs at an instant in time and then the zones immediately begin to diffuse and disperse. The diffusion takes place as the samples move through the sample chamber to the detector. This makes detection of the various zones more difficult and less accurate than may be desired. Isotachophoresis has the advantage that the zones stay relatively sharp as the sample moves through the capillary, but isotachophoresis is a difficult process to work with.
Isoelectric focusing has been employed for separation of sample components based on differences in their isoelectric points. Recently, the development of capillary electrophoresis techniques has generated interest in preforming the isoelectric focusing in capillaries, since efficient dissipation of Joule heat from a 10-100 .mu.m diameter capillary eliminates convection effects which occur in larger sample chambers and enables highly efficient separations. Capillaries with microbores, i.e., with very small inner diameter, also require only small amounts of sample, which is desirable for analysis of biological materials, such as monoclinal antibodies and other proteins. The capillary isoelectric focusing process involves establishing an electrical field between the ends of the capillary and establishing a stable pH gradient inside the capillary using a mixture of amopholytes. At the same time, an ampholytic analyte, such as a protein, moves along this pH gradient and is focused at the point where the pH is equivalent to its isoelectric point. The migration then ceases. Thus, a stationary condition is reached and maintained in the capillary. During this separation process, narrow Gaussian bands are generated with high peak concentrations which results in high separation resolution of the analytes. In order to detect the now focused analytes with available detectors, the focused zones must be moved through a stationary detector which is usually located at one end of the capillary. Thus, the focusing of the sample in the capillary is followed by a mobilization process. The commonly used mobilization process requires addition of salt to the electrolyte at one end of the capillary. The salt causes changes in pH at that end of the capillary. Because of this pH shift, the analytes focused in the capillary are no longer at their isoelectric points and will consequently move or migrate toward the end of the capillary and will pass through the detector. During the mobilization process, distortion of the focused zones and loss in resolution are unavoidable. Further, the mobilization process also takes at least about 15 minutes compared to the about five minutes required for the focusing itself using commonly available isoelectric focusing systems. Since the detection is necessary, the required mobilization makes the isoelectric focusing a relatively slow separation method thought to have little advantage compared to other capillary electrophoretic techniques. Therefore, it is necessary to develop an on-line detection method to eliminate the mobilization step and thereby improve the speed and performance of detection using the isoelectric focusing separation technique.
Several on-line scanning spectroscopic and radiometric detection methods have been developed for electrophoresis performed on slabs. However, such methods cannot be satisfactorily used with electrophoresis carried out in microbore capillaries because of their small size. Recently, there have been attempts made to continuously monitor capillary isoelectric focusing separation. In one instance, photographs were taken of the focusing of blue dye stained proteins inside a 0.4-0.6 mm i.d. capillary, and the photographs used to study the zones of proteins. However, this technique requires labeling of the proteins and can not give good quantitative information. In another instance, the separation in the capillary was monitored using chemical electrodes spaced along the length of the sample chamber. Although a complicated 100 chemical electrode array was used, the resolution obtained in these experiments was very poor.
With currently available capillary electrophoresis equipment, the capillary tube is generally about a meter in length and each end must be manually positioned in a container holding solute or sample to be separated. The longer the capillary tube, the longer the time necessary for a sample to move through the tube. When a new sample is to be separated, one end of the capillary tube has to be moved to another container which contains the new sample. Also, as the ends of the capillary are moved from container to container, the electrodes necessary for operation of the system must also be moved. Since voltages up to about 10 KV are required to operate the system, the person moving the capillary tube and electrodes from container to container may easily come into dangerous contact with the electrodes.
One of the most promising applications for capillary electrophoresis is for routine analysis in research laboratories, pharmaceutical manufacturing facilities, and hospitals. However, in many cases, relatively rapid separation and accurate detection of samples is required, because the feedback of the analyzed data is essential for observing effectiveness of a therapy, adjusting drug doses in treatment of patients in hospitals, or controlling process conditions in industrial manufacturing. Also, since different methods of capillary electrophoresis apply to different types of samples and situations, it would be convenient to be able to run different methods on the same instrument. It is impossible for current commercial capillary electrophoresis instruments to change from one separation method to another. Each instrument and capillary cartridge is designed for a particular type of separation, e.g., for capillary isotachophoresis, or for moving boundary capillary electrophoresis. A further problem is that current commercially available capillary electrophoresis instruments lack sensitive, universal, and inexpensive detectors. Although conventional absorption spectrophotometric detectors can be universal, they are not sensitive enough for capillary electrophoresis using narrow capillaries, and an expensive monochromator is required. The fluorometric detectors in use not only need expensive lasers and photomultipliers but also require fluorescent derivatization for most analytes. The commercial capillary electrophoresis instruments with such detectors are usually expensive and large devices.
Also, it is sometimes desirable to separate and identify sample components or determine if such components are present in a sample, for components which do not lend themselves to separation by electrophoretic techniques. Thus, other more complicated detectors and detection methods are required to detect these components.